Diagnostic cell imaging uses methods whereby molecules produced by cells or tissues can be specifically localized to those cells or tissues. This provides a researcher with information as to the sites of production or activity of those given molecules. For the specific localization of proteins in routine pathology, a procedure known as immunohistochemistry (IHC) is routinely utilized. In IHC an antibody for a specific antigen is applied to a fixed tissue sample that recognizes a known specific molecule as a first step. This antibody is then detected with the use of a secondary antibody that has been chemically coupled with an enzyme, such as horseradish peroxidase. After incubation with a chromogenic substrate such as di-amino-benzidine (DAB), a colored deposit is produced at the site of the secondary antibody that has bound to the primary antibody at the specific site of the protein of interest. This procedure is currently utilized for over 200 antibodies in clinical pathology labs and many more in the research environment.
The nature of tissue processing requires that the samples be “fixed” prior to embedding in paraffin and micro-sectioning on a microtome to produce tissue sections suitable for immunostaining. During this process, proteins are preserved using a formaldehyde treatment that produces chemical cross-linking which preserves the cellular features of the tissue. Formaldehyde preserves or fixes tissue or cells predominantly by cross-linking primary amine groups in proteins with other nearby nitrogen atoms in protein or DNA through a —CH2— linkage. The process of tissue fixation however, frequently masks antigens on specific proteins for which detection is desirable for diagnostic and prognostic purposes. Typically procedures are optimized for detection of individual target molecules, and when needed, serial sections are processed in a different manner for the detection of additional targets molecules. With advances in the ability to detect multiple antigens in a single sample, there needs to be a uniform tissue processing that is compatible with the detection of multiple proteins.